PROJECT SUMMARY/ABSTRACT While immunotherapy has shown great promise for robust clinical responses in renal cell carcinoma (RCC), only 1 in 3 patients will see benefit. Current clinical biomarkers, such as mutation burden or PD-L1 expression, have proven to have no predictive capacity in RCC; however, the applicant has recently identified a gene signature of DNA replication stress response (RSR) defects that has predicted immunotherapy response in multiple independent cohorts. This proposal will test the central hypothesis that RSR defects lead to activation of STING signaling to promote sensitivity to immune checkpoint blockade, and that pharmacological induction of RSR defects can sensitize otherwise resistant tumors to immunotherapy. During the K99 mentored phase, the applicant will use highly multiplexed imaging mass cytometry to analyze differences in the immune microenvironment between RSRD-low and RSRD-high patients (Aim 1.1). The applicant will then generated a diverse array of genetically engineered RCC mouse transplant models to enable controlled in vivo studies, and validate that RSR defects are causally linked to immunotherapy response (Aim 1.2). In Aim 2, the applicant will seek to mechanistically understand how RSR defects contribute to immunotherapy response by CRISPR- mediated deletion of STING from an RSRD-high model (Aim 2.1) and in vivo CRISPR screens targeting genes contained within the RSRD signature to ascertain causality (Aim 2.2). The applicant will transition to the R00 independent phase as the CRISPR screen is completed. The CRISPR screen results can serve as a basis for a first R01 submission. Aim 3 will seek to identify pharmacological compounds that will induce RSR defects to sensitize otherwise resistant models to immunotherapy, and will serve as the basis for a second R01 application. This proposal brings together an advisory committee of phenomenal researchers with expertise in DNA damage, immuno-oncology, renal cell carcinoma medical oncology, genetically engineered mouse models, and high- throughput screening to bolster the applicant's skillset in these areas. This will be supplemented by coursework in immunology and responsible conduct of research, as well as additional training to prepare the applicant for leading an independent lab. The environment at MD Anderson, including both the researchers and facilities, makes this one of the few locations where this project could be accomplished. Taken together, this proposal will equip the applicant with the tools to launch by career as a tenure-track faculty member while providing critical insight into immunotherapy for treatment of RCC to advance MD Anderson's goal of Making Cancer History.